Research suggests that vascular endothelial growth factor (VEGF) levels in the synovial fluid of knee osteoarthritis (KOA) patients are positively correlated with KOA severity. The relationship between synovial VEGF levels and pain in human KOA patients is not fully understood, and the role of VEGF in the pain pathway remains unclear. We harvested synovial membrane (SM) from 102 patients with radiographic evidence of KOA (unilateral Kellgren/Lawrence [K/L] grade 2–4) during total knee arthroplasty. Patients scored their pain on a 0 to 10 cm visual analog scale (VAS). VEGF levels in the SM of KOA patients with strong/severe (VAS ≥ 6) and mild/moderate pain (VAS < 6) were compared. Correlations between VAS and VEGF mRNA expression were investigated. To investigate a possible mechanism for VEGF-induced pain, the distribution of VEGF and the neuropeptide apelin was determined by immunohistochemical analyses. To investigate the role of VEGF in regulating apelin expression, SM cells were exposed to VEGF. VEGF expression in the VAS ≥ 6 group was significantly greater than expression in the VAS < 6 group. Expression levels of VEGF were also positively correlated with VAS. VEGF-positive cells were identified in the lining of the SM. Expression of apelin mRNA and protein were significantly elevated in SM cells treated with exogenous VEGF compared to those treated with vehicle. Synovial VEGF may be involved in pain pathways in KOA and its action may be mediated by apelin.

Uterine carcinosarcoma (UCS) represents a true example of cancer associated with epithelial-mesenchymal transition (EMT), which exhibits cancer stem cell (CSC)-like traits. Both Sox and β-catenin signal transductions play key roles in the regulation of EMT/CSC properties, but little is known about their involvement in UCS tumorigenesis. Herein, we focused on the functional roles of the Sox/β-catenin pathway in UCSs. EMT/CSC tests and transfection experiments were carried out using three endometrial carcinoma (Em Ca) cell lines. Immunohistochemical investigation was also applied for a total of 32 UCSs. Em Ca cells cultured in STK2, a serum-free medium for mesenchymal stem cells, underwent changes in morphology toward an EMT appearance through downregulation of E-cadherin, along with upregulation of Slug, known as a target gene of β-catenin. The cells also showed CSC properties with an increase in the aldehyde dehydrogenase (ALDH) 1high activity population and spheroid formation, as well as upregulation of Sox4, Sox7, and Sox9. Of these Sox factors, overexpression of Sox4 dramatically led to transactivation of the Slug promoter, and the effects were further enhanced by cotransfection of Sox7 or Sox9. Sox4 was also able to promote β-catenin-mediated transcription of the Slug gene through formation of transcriptional complexes with β-catenin and p300, independent of TCF4 status. In clinical samples, both nuclear β-catenin and Slug scores were significantly higher in the sarcomatous elements as compared to carcinomatous components in UCSs, and were positively correlated with Sox4, Sox7, and Sox9 scores. These findings suggested that Sox4, as well as Sox7 and Sox9, may contribute to regulation of EMT/CSC properties to promote development of sarcomatous components in UCSs through transcriptional regulation of the Slug gene by cooperating with the β-catenin/p300 signal pathway.

Calcitonin gene-related peptide (CGRP) is a 37-amino-acid vasodilatory neuropeptide that binds to receptor activity-modifying protein 1 (RAMP1) and the calcitonin receptor-like receptor (CLR). Clinical and preclinical evidence suggests that CGRP is associated with hip and knee joint pain; however, the regulation mechanisms of CGRP/CGRP receptor signaling in synovial tissue are not fully understood. Synovial tissues were harvested from 43 participants with radiographic knee osteoarthritis (OA; unilateral Kellgren/Lawrence (K/L) grades 3–4) during total knee arthroplasty. Correlationships between the mRNA expression levels of CGRP and those of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and cycloxygenase-2 (COX-2) were evaluated using real-time PCR analysis of total RNA extracted from the collected synovial tissues. To investigate the factors controlling the regulation of CGRP and CGRP receptor expression, cultured synovial cells were stimulated with TNF-α, IL-1β, IL-6, and prostaglandin E2 (PGE2) and were also treated with PGE2 receptor (EP) agonist. CGRP and COX-2 localized in the synovial lining layer. Expression of COX-2 positively correlated with CGRP mRNA expression in the synovial tissue of OA patients. The gene expression of CGRP and RAMP1 increased significantly in synovial cells exogenously treated with PGE2 compared to untreated control cells. In cultured synovial cells, CGRP gene expression increased significantly following EP4 agonist treatment, whereas RAMP1 gene expression increased significantly in the presence of exogenously added EP1 and EP2 agonists. PGE2 appears to regulate CGRP/CGRP receptor signaling through the EP receptor in the synovium of knee OA patients.

Recent studies have suggested that the tumor necrosis factor-α (TNF-α) pathway is a potential target for the management of osteoarthritis (OA). Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is essential in several cytokine-mediated cascades, including the TNF-α, interleukin-1 (IL-1), and TGF-β pathways. The role of TAK1 in synovial tissue in OA is not fully understood. Using synovial cells harvested from OA patients during surgery, we investigated whether TAK1 inhibition suppresses production of TNF-α-induced extracellular matrix degrading enzymes and expression of pain-related molecules. Synovial tissues were harvested from ten subjects with radiographic evidence of osteoarthritis (OA) during total knee arthroplasty. Synovial cells were cultured and stimulated with control (culture media), 10 ng/mL human recombinant TNF-α, or 10 ng/mL TNF-α and 10 μM of the TAK1 inhibitor (5Z)-7-oxozeaenol for 24 h. Real-time polymerase chain reaction (PCR) analysis was used to monitor expression of mRNA of the extracellular matrix degrading enzymes matrix metalloproteinase-3 (MMP-3) and a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 4 (ADAMTS-4); and of the pain-related molecules cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), and nerve growth factor (NGF). MMP-3 and NGF protein concentrations in cell supernatant were measured by enzyme-linked immunosorbent assay (ELISA). COX-2, mPGES-1 and ADAMTS-4 protein expression was also evaluated by western blotting. TNF-α stimulated increases in ADAMTS-4 and MMP3 mRNA (2.0-fold and 1.6-fold, respectively, p < 0.05) and protein expression (21.5-fold and 2.0-fold, respectively). Treatment with the TAK1 inihibitor (5Z)-7-oxozeaenol reduced ADAMTS-4 and MMP3 mRNA (0.5-fold and 0.6-fold, respectively) and protein expression (1.4-fold and 0.5-fold, respectively) in OA synovial cells. COX-2, mPGES-1 and NGF mRNA (11.2-fold, 3.1-fold and 2.7-fold, respectively) and protein expression (3.0-fold, 2.7-fold and 2.2-fold, respectively) were increased by TNF-α. (5Z)-7-oxozeaenol treatment reduced mPGES1 and NGF mRNA (1.5-fold and 0.8-fold, respectively) and protein (1.5-fold and 0.5-fold, respectively). TAK1 plays an important role in the regulation of TNF-α induced extracellular matrix degrading enzymes and pain-related molecule expression. TAK1 may be a potential target for therapeutic strategies aimed at preventing osteoarthritis progression and pain.

Background: Glioblastoma (GBM) is the most aggressive form of brain tumor and has vascular-rich features. The S100A4/non-muscle myosin IIA (NMIIA) axis contributes to aggressive phenotypes in a variety of human malignancies, but little is known about its involvement in GBM tumorigenesis. Herein, we examined the role of the S100A4/NMIIA axis during tumor progression and vasculogenesis in GBM. Methods: We performed immunohistochemistry for S100A4, NMIIA, and two hypoxic markers, hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase 9 (CA9), in samples from 94 GBM cases. The functional impact of S100A4 knockdown and hypoxia were also assessed using a GBM cell line. Results: In clinical GBM samples, overexpression of S100A4 and NMIIA was observed in both non-pseudopalisading (Ps) and Ps (-associated) perinecrotic lesions, consistent with stabilization of HIF-1α and CA9. CD34(+) microvascular densities (MVDs) and the interaction of S100A4 and NMIIA were significantly higher in non-Ps perinecrotic lesions compared to those in Ps perinecrotic areas. In non-Ps perinecrotic lesions, S100A4(+)/HIF-1α(−) GBM cells were recruited to the surface of preexisting host vessels in the vascular-rich areas. Elevated vascular endothelial growth factor A (VEGFA) mRNA expression was found in S100A4(+)/HIF-1α(+) GBM cells adjacent to the vascular-rich areas. In addition, GBM patients with high S100A4 protein expression had significantly worse OS and PFS than did patients with low S100A4 expression. Knockdown of S100A4 in the GBM cell line KS-1 decreased migration capability, concomitant with decreased Slug expression; the opposite effects were elicited by blebbistatin-dependent inhibition of NMIIA. Conclusion: S100A4(+)/HIF-1α(−) GBM cells are recruited to (and migrate along) preexisting vessels through inhibition of NMIIA activity. This is likely stimulated by extracellular VEGF that is released by S100A4(+)/HIF-1α(+) tumor cells in non-Ps perinecrotic lesions. In turn, these events engender tumor progression via acceleration of pro-tumorigenic vascular functions.